Method of purification of myxovirus vaccine



United States Patent 3,514,374 METHOD OF PURIFICATION OF MYXOVIRUSVACCINE William J. McAleer, Ambler, and Edward H. Wasmnth, Telford, Pa.,assignors to Merck & Co., Inc., Rahway, NJ a corporation of New JerseyNo Drawing. Filed Apr. 2, 1968, Ser. No. 718,190 Int. Cl. C12k 1/00,5/00, 7/00 US. Cl. 195-15 3 Claims ABSTRACT OF THE DISCLOSURE Prior toultracentrifuge sedimentation of myxovirus virus the growth mediumcontaining it is successively passed through 1, a filter having largepores, 2, a filter having 0.8 to 2.0 micron pores and 3, a filter having0.22 to 0.6 micron pores.

This invention relates to the production of influenza and othermyxovirus vaccines and particularly to a process for recovering thevirus from a growth medium in substantially pure form and in a highyield.

Myxovirus are influenza, para-influenza, measles and mumps. They aredistressing causes of both severe and mildly acute illnesses in man. Toreduce the incidence of these illnesses, vaccines made from the killedviruses are available but they have the disadvantage of being expensiveto make in highly purified form. The virus to be used in making thevaccine is grown in living cells such as in the allantoic fluid cells ofeggs or in culture media containing animal tissue cells and thereafterthe virus must be recovered in the purest possible form, free of theculture cells and constituents thereof. Previous vaccines haveheretofore been available only in highly impure state and the impurities(such as egg protein) can cause severe reaction in persons sensitive tothese materials.

Prior processes for the recovery of a relatively purified virus havebeen essentially batch operations involving cumbersome and complexmanual manipulations. Moreover, prior processes did not specificallyremove microbial contaminants. A known process utilizesadsorption-desorption techniques and although it produces a fairly purevirus in a relatively high yield, it is time-consuming and thereforeresults in an expensive product. A reduction in the cost of the purifiedvirus is desirable as it increases the number of persons who can affordthe purchase of the vaccine which is so important to their health.

T he present invention provides a process which includes substantiallycontinuous operational steps and these operations can readily be carriedon in large scale factory flow systems. Not only is a highly purifiedvirus obtained but it is produced in a high yield in a relatively shorttime and consequently an inexpensive vaccine can be made.

The process of this invention employs the conventional use of anultracentrifuge, e.g. one operating above 50,000 g, but the feature ofthe invention is the use of filtration steps just prior to thisultracentrifugation to remove contaminants which are larger than thevirus and principally microbial contaminants. These pretreatments beforeultracentrifugal sedimentation make possible the direct preparation ofhighly purified, sterile vaccines directly from the centrifuged productswithout subsequent treatments. The initially sedimented vaccines of theprior art are substantially less pure and nonsterile.

Prior to the present invention it was thought that it would beimpossible to employ preliminary filtration of the culture mediumcontaining the virus, prior to centrifugation for the reason that thefilter pads would promptly clog up and prevent the passage of the virus.It was thought that if large pore filtration pads were used to overcomeloss of virus on the pads, too much of the coarse contaminants wouldpass through the filter.

The invention involves the known use of an ultracentrifuge to sedimentthe virus from a liquid containing it and the feature of the inventionis the use of specific filtration conditions which are carried on justprior to the ultracentrifugation step. The invention involves thediscovery that the successive use of filters of specified pore size,their large surface area, and an established flow-rate through them,will produce a liquid containing nearly all the virus which isessentially free of contaminatory substances from the growth medium.This end filtrate can be delivered directly to the ultracentrifuge toobtain the virus in concentrated form. This concentrated virus is to beused in making a vaccine by resuspending it in a conventional manner inan injectable liquid such as physiological phosphate buffer.

The specific details of the filtration step of this invention will bedefined but it should first be mentioned that prior to the filtrationoperation, it is best but not essential that a conventionalclarification step be performed. This preliminary clarification shouldremove relatively large particles of the culture media, such as cellularclumps from the allantoic fluid in which the virus Was grown.

For this purpose a moderate speed, disc-type centrifuge (such as DeLavalModel PX) operating in the general field of 15,000 g is suitable. It isto be understood that other preliminary gross clarification methods maybe used, such as ordinary settling and decanting, and that in fact thispreliminary clarification may be eliminated altogether. Its elimination,however, will require more frequent change of the subsequent initialfiltration pad.

The filtration steps of this invention are specifically established tosuccessively remove smaller and smaller microbial and othercontaminating particles so that the filtrate is a sterile liquidcontaining only the virus particles. This filtration involves thefollowing sequence steps:

(a) Relatively large pore prefilter such as cellulosic pads,diatomaceous earths, or spun glass. (Millipres Spef. Apr.)

(b) 1.2 micron pad preferably, but can be in the 0.8-

2.0 micron range.

(c) 0.45 micron pad preferable, but can be in the 0.220.6

range.

It is important that for each filtration step the pad area be aboutsquare centimeters for each 1 to 1.5 liters of the fluid and that forevery 100 square centimeters the flow rate be in the range of 1 to 1.5liters per 10 minutes. To speed up this filtration operation, if itproves to be necessary to obtain this flow rate, air pressure may beapplied to one or all three filtration steps. With increased fluidvolume above this range some plugging of the filter can occur and thiswill result in failure of some virus to pass through. It is importantthat the filtration area be relatively large as specified above asotherwise the operation will be slow, complete filtration at each stepwill not be achieved and loss of virus on the pads will occur.

The final filtrate from these steps will be found to be clear andsterile and to contain nearly all of the original virus growth. Tosediment the virus particles in this filtrate the high speed centrifugeis utilize and this may be in the 50,000 to 80,000 g field. Arepresentative apparatus is the Sharples Model T-1P Presurtite. Thiswill concentrate the virus to a thick slurry and this operation shouldbe carried out as a continuous operation. For commercial production itwould be preferable to use a zonal type ultracentrifuge such as theSpinco Md L-4 Zonal Centrifuge or the Oak Ridge K11 Zonal Centrifuge.

This final concentrated virus from the centrifuge may be used at once tomake a vaccine by adding it to an injectable liquid to a suitabledilution, or the slurry may be put up in sterile form, afterlyophilization, if desired,

for subsequent resuspension.

The virus may be killed at any step in the operation. This may be beforethe initial clarification if it is used, during one of the filtrationsteps or after ultracentrifugation. Known inactivation agents such asFormalin may be used, the amount and concentration being in accordancewith established standards.

Although the invention will be described in the examples with particularreference to influenza virus which has been grown in allantoic fluid andhas subsequently been killed, the invention may be practiced with otherviruses, grown in other culture medium such as ones containing tissuecells, and the virus may or may not be killed.

Representative examples are the following:

EXAMPLE 1 450 fertilized hens eggs were infected with A strain ofinfluenza (JAP 170) and held at 37 C. for 40 hours. The allantoic fluidcontaining the grown virus was removed from the eggs by suction andcollected into a pool measuring about 4500 ml. The fluid was thentreated with paraformaldehyde at the 4 by volume level at 35 C. for 24hours, to inactivate the virus.

The entire pool was run at 300 ml./min. through De Laval Gyro Tester orsimilar centrifuge operating at 12,000 rpm. (about 15,000 g) and theclarified fluid was collected.

Then centrifuge-clarified fluid was first passed through a 500 cm. spunglass prefilter such as a No. 142 Millipore filter, next through a 500cm. l.2 micron filter pad and finally through a 500 cm. 0.45 micronfilter pad. Air pressure of about p.s.i. was applied at each step andthe total time of filtration was 8 minutes. Recovery of the vaccine atthis state is 86% based on standard CCA assays.

Final concentration of the vaccine was accomplished by sedimentation ina high speed centrifuge operating at 50,000 r.p.m. with a fluid flow atabout 1.0 liter per hour. The sedimented virus is used to make thevaccine.

EXAMPLE 2 3500 fertilized hens eggs were infected with a B strain ofinfluenza (B/rnass) and held at 37 C. for 40 hours. The allantoic fluidwas removed from the eggs by suction and collected into a pool measuringabout 35,000 mls. The fluid was treated with formaldehyde at the byvolume level at 35 C. for 24 hours.

The entire pool was run at 300 mls./min. through a centrifuge such as aDe Laval Gyro Tester operating at 12,000 r.p.m. Air pressure was used toforce the liquid through the centrifuge and the rate was measured with aflowmeter to approximately maintain the rate. The clarified fluid wascollected in a pressure vessel.

This centrifuge-clarified fluid was first forced through a 3100 cm.prefilter pad (Multiplate Filter Holder) followed by a 3100 cm. 1.2filter pad and finally through a 2300 cm. 0.45;]. filter pad. Thefiltration air pressure used was 20 psi. and the total time offiltration was 15 minutes. Recovery of the vaccine at this stage is 84%based on a standard CCA assay.

Final concentration of the vaccine was accomplished in a SharplesHi-Speed Centrifuge operating at 50,000 r.p.m. with a fluid flow of 1.5l./ hr. The sedimented virus is used to make the vaccine.

EXAMPLE 3 The process of either of the above examples is carried outwith the change that the hens eggs are infected with one of the viruses,para-influenza, measles or mumps.

In the above examples the initial centrifugal clarification can beomitted and the initial filter pad be replaced more often.

What is claimed is:

1. A method of obtaining sterile, sedimented myxovirus from eggallantoic fluid growth medium which comprises:

(A) centrifugation at about 15,000 g;

(B) passing up to 1.5 liters of the growth medium per cm. of filter areaat a rate of 100 to ml./ minute/100 cm. of filter area successivelythrough filter pads having the determined porosity of:

(l) a prefilter material selected from the group consisting ofcellulose, diatomaceous earth, and spun glass,

(2) 0.8 to 2.0 micron pores,

(3) 0.22 to 0.6 micron pores, and

(C) centrifuging the filtrate in a 50,000 to 89,000 g field.

2. The method according to claim 1 wherein filters (2) and (3) haveporosities of 1.2 and 0.45 microns respectively.

3. The method according to claim 1 wherein the myxovirus is an influenzavirus.

References Cited UNITED STATES PATENTS 8/1944 Rake et al. 424-89 X9/1958 Novak 210-335

